Distinct cytokine profiles in malaria coinfections: A systematic review

Background Few data exist on the distinct cytokine profiles of individuals with malaria coinfections and other diseases. This study focuses on data collation of distinct cytokine profiles between individuals with malaria coinfections and monoinfections to provide evidence for further diagnostic or prognostic studies. Methods We searched five medical databases, including Embase, MEDLINE, PubMed, Ovid, and Scopus, for articles on cytokines in malaria coinfections published from January 1, 1983 to May 3, 2022, after which the distinct cytokine patterns between malaria coinfection and monoinfection were illustrated in heat maps. Results Preliminary searches identified 2127 articles, of which 34 were included in the systematic review. Distinct cytokine profiles in malaria coinfections with bacteremia; HIV; HBV; dengue; filariasis; intestinal parasites; and schistosomiasis were tumor necrosis factor (TNF), interferon (IFN)-γ, IFN-α, interleukin (IL)-1, IL-1 receptor antagonist (Ra), IL-4, IL-7, IL-12, IL-15, IL-17; TNF, IL-1Ra, IL-4, IL-10, IL-12, IL-18, CCL3, CCL5, CXCL8, CXCL9, CXCL11, granulocyte colony-stimulating factor (G-CSF); TNF, IFN-γ, IL-4, IL-6, IL-10, IL-12, CCL2; IFN-γ, IL-1, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, CCL2, CCL3, CCL4, G-CSF; IL-1Ra, IL-10, CXCL5, CXCL8, CXCL10; TNF, IL-2, IL-4, IL-6, IL-10; and TNF, IFN-γ, IL-4, IL-5, IL-10, transforming growth factor-β, CXCL8, respectively. Conclusion This systematic review provides information on distinct cytokine profiles of malaria coinfections and malaria monoinfections. Further studies should investigate whether specific cytokines for each coinfection type could serve as essential diagnostic or prognostic biomarkers for malaria coinfections.

Introduction family. M-CSF and G-CSF are lineage-specific cytokines that affect macrophage and neutrophil survival, growth, differentiation, and function, respectively [16].
In malaria-endemic areas, malaria can coinfect with other tropical diseases as they have overlapping geographical distributions. Notably, several malaria coinfections with other tropical diseases have been documented, including dengue [17], hookworm [18], human African trypanosomiasis [19], typhoidal/nontyphoidal Salmonella [20], scrub typhus [21], visceral leishmaniasis [22], leptospirosis [23], Chikungunya [24], and the most recent coronavirus disease (COVID- 19) or severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) [25]. Because malaria coinfections may lead to severe diseases, such as malaria-dengue [26], malaria-human African trypanosomiasis [19], or malaria-nontyphoidal Salmonella coinfections [20], studies on malaria coinfections are crucial for understanding the role of other diseases in malaria pathophysiology and clinical outcomes. Although distinct cytokine profiles of malaria infection have been intensively examined over the last decade, studies on distinct cytokine profiles of individuals with malaria coinfections with other diseases remain underexplored. Therefore, this study focuses on data collation of distinct cytokine profiles of individuals with malaria coinfections to provide insights into the current status of cytokine studies and identify research gaps regarding whether cytokines can serve as disease markers or be used to track the progression of concurrent infections.

Search strategy and selection criteria
This study was registered with PROSPERO, CRDCRD42022331608. For this systematic review and meta-analysis, we adhered to the protocol outlined in the updated PRISMA 2020 guideline (S1 PRISMA Abstract Checklist; S1 PRISMA 2020 Checklist) [27]. We searched five medical databases (Embase, MEDLINE, PubMed, Ovid, and Scopus) for articles on cytokines in malaria coinfections published from January 1, 1983, to May 3, 2022. We also searched Google Scholar and the reference lists of the included studies and reviews for relevant studies. The search terms and complete eligibility requirements are detailed in the S1 Table. The search was restricted to articles written in English. Medical subject heading terms were used in the PubMed search, and their synonyms were modified for each database. For study selection, original papers reporting cytokines in malaria coinfections versus malaria monoinfections would be examined for eligibility. Meanwhile, animal studies, in vitro studies, reviews, conference abstracts, comments, and letters were excluded. We used EndNote version 20 software (Clarivate Analytics, Philadelphia, PA, USA) to remove duplicate articles and manually check the remaining for duplicates. The abstract screening and full-text selection were conducted independently by two authors (MK and KUK) following predetermined eligibility criteria (S1 Table). If the study selection was not unanimously agreed upon, a third author (WM) resolved this disagreement by making a final selection decision. For inclusion in meta-analysis, studies must report the mean and standard deviation (or median and interquartile range) of cytokines in malaria coinfections and monoinfections. In addition, the cytokines included in the meta-analysis must be reported in two or more studies, which is the requirement for a meta-analysis [28].

Data extraction and quality assessment
Data extraction was performed using a standardized data extraction form. For cytokine levels reported in at least two studies, we extracted the mean (also standard deviation) or median (also interquartile range) from studies reporting cytokines in coinfections and malaria monoinfections. Only the study with the largest sample size was included if two or more articles reported the same setting and participants. The JBI's critical appraisal tools were utilized to evaluate the studies' quality and risk of bias. Two independent reviewers (KUK and WM) evaluated the quality of the included studies. Disagreements were resolved through discussion. Using Microsoft Excel 365, data extraction and study quality assessment were performed.

Data syntheses
For qualitative synthesis, we illustrated the distinct cytokine patterns between malaria coinfection and malaria monoinfection in heat maps. Red, green, and yellow colors mean increased, comparable, and decreased levels of cytokines between the two groups, respectively. The distinct cytokine patterns between the two groups were then described in narrative form. For quantitative synthesis, we performed meta-analysis to combine the effect sizes of the mean difference (MD) of cytokine levels between malaria coinfections and malaria monoinfections using Stata version 17.0 (Stata Corporation, College Station, TX). The pooled estimates were calculated using random-effect models because all populations were assumed to be distinct. The DerSimonian-Laird method was used to estimate the variance between studies. In addition, subgroup analyses of coinfection types (bacteremia, viruses, and other parasites) were performed to identify cytokine differences unique to those populations. If mean and standard deviation were not reported by studies, the mean and standard deviation were calculated from the median and interquartile range as suggested elsewhere [29]. If studies reported the mean and standard deviation in several groups, we also combined means and standard deviation into one group, as described previously [30]. Using the Cochran Q statistic and I 2 statistic, heterogeneity between studies was measured. Studies were deemed heterogeneous when the P value for the Cochran Q statistic was less than 0.05; the heterogeneity levels were classified as low (I 2 < 25 percent), low to moderate (25% to 50%), moderate to high (50% to 75%), or high (>75%) [31]. To determine if the pooled estimates were robust, sensitivity analysis (leave-oneout method) was performed in which studies with extreme effect sizes and heterogeneity were excluded [32]. Using Egger's test, the small-study effect was evaluated to detect publication bias. When the p value of Egger's test was lower than 0.05, publication bias was suspected.

Results
Our initial searches identified 2127 articles, of which 34 were included in the systematic review (Fig 1).
Notably, 82% (28 of 34) of the included studies were conducted in Africa and published between 2004 and 2022; 47% (16 of 34) were conducted using a cross-sectional study design; 23.5% (8 of 34) were prospective observational studies; and the remaining 29.4% (10 of 34) were cohort, case-control, retrospective observational, and clinical trial studies. Approximately 79.4% (27 of 34) of the studies enrolled patients infected with P. falciparum, and the remaining studies enrolled patients infected with other species. While 52.9% (18 of 34) of the studies reported malaria coinfections with other parasites, 38.2% (13 of 34) reported malaria coinfections with viruses. Table 1 provides a summary of the included studies, whereas S2 Table presents the details of the studies included in the systematic review and meta-analysis. S3 Table  presents the clinical characteristics, coinfection types, and age groups of each study. All 34 studies were assessed for their quality (S4 Table) and were included in the systematic review.
Among the 34 studies included in the systematic review, we identified 35 cytokines tested for their distinct expression in malaria coinfections compared to malaria monoinfections.     Distinct cytokine profiles in malaria and HBV coinfections were TNF, IFN-γ, IL-4, IL-6, IL-10, IL-12, and CCL2. IFN-γ, IL-10, and CCL2 levels were significantly increased in coinfections compared to malaria monoinfection, as observed by Cruz et al. [41]. TNF levels were significantly increased in asymptomatic malaria and HBV coinfections, but their levels were significantly decreased in uncomplicated malaria and HBV coinfections [41]. IL-10 levels were increased, as observed by Cruz et al.
According to van den Bogaart et al. [51], the coinfection group had significantly higher TNF and IFN-γ levels than the malaria monoinfection group.

Discussion
The present study showed distinct cytokine profiles in malaria coinfections compared to malaria monoinfections. Malaria coinfections with bacteremia were characterized by distinct cytokine profiles, including elevated IFN-γ, IFN-α, IL-4, IL-7, IL-12, IL-15, and IL-17 levels and decreased TNF levels, indicating a strong cytokine response. This strong cytokine response caused by malaria and bacteremia coinfections could result in worse clinical ND: Not determined due to no study for analysis or the number of studies for analysis were less than three. https://doi.org/10.1371/journal.pntd.0011061.t002 outcomes [66]. Bacteremia coinfections with malaria could enhance the clinical severity of anemia and sepsis by decreasing cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) expression [33], and these mediators were associated with TNF-α, IFN-γ, and IL-10 production [67,68] in coinfected individuals. Notably, proinflammatory cytokines were found to be highest in Gram-negative bacteremia coinfections, followed by Gram-positive bacteremia coinfections, and lowest in malaria monoinfections, suggesting that this cytokine profile promotes parasite clearance [34]. In bacteremia coinfections, one of the proinflammatory cytokines, such as TNF, was decreased. Through a feedback mechanism, the decreased TNF levels in coinfected patients may be due to the counterregulatory activities of IFN-induced increased nitric oxide (NO) that downregulate nitric oxide synthase (NOS)-inducing TNF-α [69]. Davenport et al. [34] reported decreased TNF levels in malaria and bacteremia coinfections, but another study [33] reported comparable levels of this cytokine between coinfections and malaria monoinfection, suggesting contradictory results between studies. Distinct cytokine profiles in malaria and HIV coinfections compared to malaria monoinfections were increased IL-12, CXCL9, CXCL11, IL-18, and G-CSF levels and decreased TNF and IL-4 levels. Most of the distinct cytokine profiles in malaria and HIV coinfections were chemokines that act as chemoattractant for leukocytes playing roles in malaria pathogenesis. Although the exact mechanism of these chemokines in malaria pathogenesis is unknown, increased levels of chemokines such as CXCL9, CXCL10, and CXCL11 have been proposed as predictive markers for HIV disease progression [70]. G-CSF is another chemokine that contributes to the proliferation and differentiation of neutrophil granulocytes [71]. Increased G-CSF levels were associated with severe falciparum malaria, indicating a defense mechanism against malaria parasites [72]. IL-12 is a potent immunomodulatory cytokine that increases cell-mediated and humoral immune responses to malaria parasites by inducing isotype switching [73]. IL-4 is a potent immunomodulatory cytokine that correlates with the severity of malaria hyperparasitaemia but not the severity of the disease [74]. Although TNF had been proposed as a prognostic biomarker of severe malaria, as its levels were increased in patients with severe malaria [75], this study showed that TNF levels were significantly decreased in malaria and HIV coinfections. Evidence of lower TNF levels in these coinfections remains unclear. Notably, evaluating several TNF receptors or cytokine networks rather than TNF alone could prove to be more reliable markers of TNF activity [37].
[41] explored cytokines in both asymptomatic and uncomplicated malaria and revealed differences in TNF levels in malaria coinfections compared with malaria monoinfection-TNF levels were increased in asymptomatic but decreased in uncomplicated malaria [41]. These results indicated that coinfection by HBV drives the reduction of systemic inflammation caused by malaria parasites [80]. CCL2 is a chemokine that influences CD4 + T lymphocytes to produce IL-4 cytokine [81]. Therefore, increased CCL2 levels in coinfections may indicate a modulatory effect of IL-4 to downregulate key proinflammatory cytokines such as TNF-α [82]. dengue coinfections were IFN-γ, IL-1, IL-4, IL-6,  IL-10, IL-12, IL-13, IL-17, CCL2, CCL3, CCL4, and G-CSF. Malaria and dengue coinfections showed a significant increase in proinflammatory cytokines such as IFN-γ, IL-1, IL-6, and IL-12; Th2 cytokines such as IL-4; anti-inflammatory cytokines such as IL-10 and IL-13; and Th17 cytokines such as IL-17. In addition, the levels of several chemokines, such as CCL3, CCL4, and G-CSF, also increased. Notably, both malaria and dengue have been linked to strong activation of proinflammatory cytokines and Th1 cytokines [43]. Therefore, increased levels of proinflammatory immune markers in the coinfection of malaria and dengue may cause the synergistic activation of immunological pathways of cytokines. Cases of malaria and dengue coinfection also exhibited the highest values of IFN-γ and IL-6. A study suggested that dengue viruses can affect the immune response of individuals with malaria and dengue coinfections as the number of parasitemia was lower in patients with coinfections [44]. These observations corroborated our previous findings that individuals with malaria and dengue coinfections were at a higher risk of severe diseases than those with monoinfection [26].

Distinct cytokine profiles in malaria and
The prevalence of malaria and CHIKV coinfections among febrile patients has been shown to vary with diagnostic tests for CHIKV infection, and coinfection occurred by chance [24]. The present study showed that the levels of proinflammatory cytokines such as IFN-γ and IL-12; Th2 cytokines such as IL-4; and anti-inflammatory cytokines such as IL-13 were increased in coinfections, but the levels of some proinflammatory cytokines, such as TNF and IL-12, were decreased in coinfections. These results indicated that TNF and IL-12 activities were suppressed among individuals with coinfections. Malaria monoinfection exhibited high TNF and IL-12 responses. Therefore, malaria and CHIKV coinfections might suppress viral replication [45] or inhibit the severity of parasite infection.
Malaria and other parasite coinfections demonstrated varying cytokine responses. Most of the cytokines that were investigated in malaria and other parasite coinfections were TNF, IFNγ, IL-2, IL-4, IL-6, and IL-10. TNF levels were increased in malaria coinfections with human African trypanosomiasis [49] and intestinal parasites [52,53]. Meanwhile, IFN-γ levels were increased among individuals with human African trypanosomiasis [50], schistosomiasis [52,59,60], and visceral leishmaniasis [51]. Other cytokines such as IL-4, IL-6, and IL-10 were also elevated in coinfections compared to malaria monoinfection. These results indicated that malaria and other parasite coinfections exhibited stronger proinflammatory cytokine responses than malaria monoinfections. However, a small number of studies have investigated these cytokines, and not all studies have evaluated similar cytokines in coinfections. IFN-γ levels were distinctly increased among malaria and schistosomiasis coinfections in three studies [52,59,60]. Although IFNγ can induce the production of TNF-α and other molecules, most studies have demonstrated that malaria and schistosomiasis coinfections have comparable TNF levels compared to malaria monoinfection. Therefore, other factors, such as age, intensity of infection, and hormones, could directly affect the balance between proinflammatory and anti-inflammatory cytokines in these types of coinfections [59]. IL-6 levels were decreased in most studies that investigated malaria and intestinal parasite coinfections ( [48,52,83]. IL-6 has been implicated as a candidate marker for severe malaria, as its levels were increased in severe malaria compared to uncomplicated malaria [77]. Therefore, decreased IL-6 levels in patients with malaria and intestinal parasite coinfections might be a protective factor for disease severity among individuals with coinfections. The next common cytokine that studies evaluated in both malaria and other parasite coinfections was IL-10. IL-10 levels were elevated in human African trypanosomiasis [50], hookworm and soil-transmitted helminths [54,56], and schistosomiasis coinfections [58,59,61]. Again, distinct IL-10 levels were frequently observed among schistosomiasis coinfections compared to malaria monoinfection. This result indicates that IL-10 plays a role in regulating excessive inflammatory responses by its potent anti-inflammatory cytokines and could provide protection against severe disease [84].
The meta-analysis results based on the limited number of studies showed that IFN-γ and CXCL8 levels were significantly increased in malaria coinfections compared to malaria monoinfection. Meanwhile, TNF levels were significantly decreased in malaria coinfections compared to malaria monoinfection. Some limitations exist regarding the number of studies included in the systematic review. First, because the number of studies for each malaria coinfection type was small, the conclusion of distinct cytokine profiles provided in malaria coinfections with human African trypanosomiasis, visceral leishmaniasis, and Chikungunya compared to malaria monoinfection could not be made. Second, cytokine responses in the different malaria species were not analyzed because most studies reported P. falciparum coinfections. Third, considering data heterogeneity and the small number of studies that reported cytokine levels in each coinfection type, the analysis or synthesis of cytokine levels between children and adults, or severe and uncomplicated malaria, could not be performed. Because only a few studies have reported the cytokine levels in each severe malaria complication that may exert the difference in cytokine profile between malaria coinfection and malaria monoinfection, the evidence could not be synthesized. In addition, because the data on cytokine levels in each severe malaria complication were limited, additional research is required to determine these cytokine levels in various severe malaria complications.

Conclusion
The systematic review revealed distinct cytokine profiles in malaria coinfections compared to malaria monoinfections. Further research is required to determine whether specific cytokines for each coinfection type could serve as useful diagnostic or prognostic biomarkers for malaria coinfections. Future investigations of the following factors will be crucial: IFN-α, IL-15, and IL-17 levels for malaria and bacteremia coinfections; IL-12 and CXCL9 levels for malaria and HIV coinfections; TNF and IL-10 levels for malaria and HBV coinfections; IFN-γ, IL-6, and IL-13 levels for malaria and dengue coinfections; IL-6 and IL-10 levels for malaria and intestinal parasite coinfections; and IFN-γ and IL-10 levels for malaria and schistosomiasis coinfections.